Regulators of bacterial virulence factor expression

Abstract

Genetic loci that encode polypeptides that regulate the expression of virulence factors in bacteria, particularly S. aureus . Methods of detecting the gene or gene products for purposes of detecting S. aureus or diagnosing a patient suspected of being infected by S. aureus.

We claim:  
     
         1 . An isolated polynucleotide comprising a member selected from the group consisting of: 
 (a) a polynucleotide of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 or a complement of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (b) a fragment of the polynucleotide of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 wherein the fragment comprises at least 20 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (c) a polynucleotide having at least 70% sequence identity to the sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (d) a polynucleotide that hybridizes to any one of the polynucleotides of (a), (b) or (c) under conditions of 5× SSC, 50% formamide and 42° C., and which encodes a protein having the same biological function; and    (e) a polynucleotide encoding the same amino acid sequence as any of the polynucleotides of (a), (b), (c) or (d) but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code.    
     
     
         2 . A recombinant polynucleotide comprising a member selected from the group consisting of: 
 (a) a polynucleotide of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 or a complement of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (b) a fragment of the polynucleotide of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 wherein the fragment comprises at least 20 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (c) a polynucleotide having at least 70% sequence identity to the sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (d) a polynucleotide that hybridizes to any of the polynucleotides of (a), (b) or (c) under conditions of 5× SSC, 50% formamide and 42° C., and which encodes a protein having the same biological function; and    (e) a polynucleotide encoding the same amino acid sequence as any of the polynucleotides of (a), (b), (c) or (d) but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code.    
     
     
         3 . The polynucleotide of  claim 2  wherein the polynucleotide is selected from the group consisting of: 
 (a) a polynucleotide of SEQ ID NO:3 or a complement of SEQ ID NO:3;  
 (b) a fragment of the polynucleotide of SEQ ID NO:3 wherein the fragment comprises at least 20 contiguous nucleotides of SEQ ID NO:3;  
 (c) a polynucleotide having at least 70% sequence identity to the sequence of SEQ ID NO:3;  
 (d) a polynucleotide that hybridizes to any of the polynucleotides of (a), (b) or (c) under conditions of 5× SSC, 50% formamide and 42° C., and which encodes a protein having the same biological function; and  
 (e) a polynucleotide encoding the same amino acid sequence as any of the polynucleotides of (a), (b), (c) or (d) but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code.  
 
     
     
         4 . A recombinant vector comprising a polynucleotide of  claim 2 .  
     
     
         5 . The recombinant vector of  claim 4  wherein the vector is selected from the group consisting of plasmids, bacteriophages, cosmids, and viruses.  
     
     
         6 . The recombinant vector of  claim 4  wherein the vector further comprises at least one additional sequence chosen from the group consisting of: 
 (a) regulatory sequences operatively coupled to the polynucleotide;  
 (b) selection markers operatively coupled to the polynucleotide;  
 (c) marker sequences operatively coupled to the polynucleotide;  
 (d) a purification moiety operatively coupled to the polynucleotide;  
 (e) a targeting sequence operatively coupled to the polynucleotide; and  
 (f) a sequence directing expression of a heterologous polypeptide.  
 
     
     
         7 . The recombinant vector of  claim 6  wherein the vector is selected from the group consisting of plasmids, bacteriophages, cosmids, and viruses.  
     
     
         8 . The recombinant vector of  claim 6  wherein the vector comprises a promoter selected from the group consisting of trp, lac, P L , and T7 polymerase operably coupled to the polynucleotide.  
     
     
         9 . A host cell comprising the recombinant vector of any of  claims 4  to  8 .  
     
     
         10 . The host cell as set forth in  claim 9  wherein said host cell is selected from the group consisting of mammalian cells, plant cells, insect cells, yeast, bacteria, bacteriophage.  
     
     
         11 . The host cell as set forth in  claim 9  wherein said host cell expresses a protein encoded by said vector.  
     
     
         12 . A protein or polypeptide encoded by the polynucleotide selected from the group consisting of: 
 (a) a polynucleotide of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 or a complement of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (b) a fragment of the polynucleotide of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 wherein the fragment comprises at least 20 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (c) a polynucleotide having at least 70% sequence identity to the sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;    (d) a polynucleotide that hybridizes to any of the polynucleotides of (a), (b) or (c) under conditions of 5× SSC, 50% formamide and 42° C., and which encodes a protein having the same biological function; and    (e) a polynucleotide encoding the same amino acid sequence as any of the polynucleotides of (a), (b), (c) or (d) but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code.    
     
     
         13 . An isolated protein or polypeptide comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         14 . A recombinant protein or polypeptide comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         15 . An isolated protein or polypeptide comprising a Rot polypeptide sequence from  Staphylococcus aureus , wherein the polypeptide comprises at least 15 contiguous amino acids of SEQ ID NO:2.  
     
     
         16 . A protein or polypeptide of  claim 12  wherein one or more of the amino acids have been substituted with a conserved amino acid and the biological function of the protein has been maintained.  
     
     
         17 . A protein or polypeptide of  claim 12  wherein the protein or polynucleotide is selected from the group consisting of: 
 (a) a polynucleotide of SEQ ID NO:3 or a complement of SEQ ID NO:3;  
 (b) a fragment of the polynucleotide of SEQ ID NO:3 wherein the fragment comprises at least 20 contiguous nucleotides of SEQ ID NO:3;  
 (c) a polynucleotide having at least 70% sequence identity to the sequence of SEQ ID NO:3;  
 (d) a polynucleotide that hybridizes to any of the polynucleotides of (a), (b) or (c) under conditions of 5× SSC, 50% formamide and 42° C., and which encodes a protein having the same biological function; and  
 (e) a polynucleotide encoding the same amino acid sequence as any of the polynucleotides of (a), (b), (c) or (d) but which exhibits regular degeneracy in accordance with the degeneracy of the genetic code.  
 
     
     
         18 . An isolated protein or polypeptide comprising the amino acid sequence as depicted in SEQ ID NO:4.  
     
     
         19 . A recombinant protein or polypeptide comprising the amino acid sequence as depicted in SEQ ID NO:4.  
     
     
         20 . An isolated protein or polypeptide comprising an Rlp polypeptide sequence from  Staphylococcus aureus , wherein the protein or polypeptide comprises at least 15 contiguous amino acids of SEQ ID NO:4.  
     
     
         21 . The protein or polypeptide of  claim 12  or  17  wherein one or more of the amino acids have been substituted with a conserved amino acid and the biological function of the protein has been maintained.  
     
     
         22 . An isolated, purified antibody that specifically binds to a polypeptide of  claim 12 .  
     
     
         23 . The antibody of  claim 22  wherein the antibody is a monoclonal antibody.  
     
     
         24 . An isolated, purified antibody that specifically binds to a polypeptide as set forth in  claim 17 .  
     
     
         25 . The antibody of  claim 24  wherein the antibody is a monclonal antibody.  
     
     
         26 . A fusion protein comprising a polypeptide or protein of  claim 11  linked to a heterologous protein or polypeptide.  
     
     
         27 . A nucleic acid probe or primer comprising at least 20 contiguous nucleotides of a polynucleotide of  claim 2 .  
     
     
         28 . A nucleic acid probe or primer comprising at least 20 contiguous nucleotides complementary to a polynucleotide of  claim 2 .  
     
     
         29 . A nucleic acid probe or primer comprising at least 20 contiguous nucleotides of a polynucleotide of  claim 3 .  
     
     
         30 . A nucleic acid probe or primer comprising at least 20 contiguous nucleotides complementary to a polynucleotide of  claim 3 .  
     
     
         31 . A method for detecting a polynucleotide of  claim 2  in a microbial isolate, comprising: 
 (a) extracting the DNA of a microbial isolate; and  
 (b) probing said DNA with a labeled nucleic acid probe constructed from a polynucleotide of  claim 2 .  
 
     
     
         32 . A method for detecting a polynucleotide of  claim 3  in a microbial isolate, comprising: 
 (a) extracting the DNA of a microbial isolate; and  
 (b) probing said DNA with a labeled nucleic acid probe constructed from a polynucleotide of  claim 3 .  
 
     
     
         33 . A method for detecting a protein or polypeptide of  claim 12  in a biological sample, comprising: 
 (a) combining a biological sample with an antibody of  claim 22  or  24  under conditions that permit the formation of a stable antigen-antibody complex; and  
 (b) detecting any stable complexes formed in step (a).  
 
     
     
         34 . A method for detecting a protein or polypeptide of  claim 17  in a biological sample, comprising: 
 (a) combining a biological sample with an antibody of  claim 23  or  25  under conditions that permit the formation of a stable antigen-antibody complex; and  
 (b) detecting any stable complexes formed in step (a).  
 
     
     
         35 . A method for detecting an anti- Staphylococcus aureus  Rot antibody in a biological sample, comprising: 
 (a) combining a biological sample which potentially contains an anti- Staphylococcus aureus  rot antibody with a polypeptide of  claim 12  under conditions which permit formation of a stable antigen-antibody complex; and    (b) detecting any stable complexes formed in step (a).    
     
     
         36 . A method for detecting an anti- Staphylococcus aureus  Rlp antibody in a biological sample, comprising: 
 (a) combining a biological sample which potentially contains an anti- Staphylococcus aureus  rlp antibody with a polypeptide of  claim 12  under conditions which permit formation of a stable antigen-antibody complex; and    (b) detecting any stable complexes formed in step (a).    
     
     
         37 . A method for purifying an anti- Staphylococcus aureus  Rot antibody present in a biological sample, comprising: 
 (a) combining the biological sample with a polypeptide of  claim 12  under conditions which permit formation of a stable antigen-antibody complex;    (b) separating any stable complexes formed in step (a) from said biological sample; and    (c) isolating said antibody from any stable complexes separated from the biological sample in step (b).    
     
     
         38 . A method for purifying an anti- Staphylococcus aureus  Rlp antibody in a biological sample, comprising: 
 (a) combining the biological sample with a polypeptide of  claim 12  under conditions which permit formation of a stable antigen-antibody complex;    (b) separating any stable complexes formed in step (a) from said biological sample; and    (c) isolating said antibody from any stable complexes separated from the biological sample in step (b).    
     
     
         39 . A method of screening for an antibacterial agent, comprising: 
 (a) contacting a cell expressing a polypeptide encoded by a gene selected from the group consisting of the polynucleotides as set forth in  claim 2  with a test compound; and    (b) determining whether the amount or level of activity of said polypeptide is increased.    
     
     
         40 . The method of  claim 39  wherein said increase or decrease is measured by assaying the protein level of the expressed polynucleotide.  
     
     
         41 . The method of  claim 39  wherein said increase or decrease is measured by assaying the RNA level of the expressed polynucleotide.  
     
     
         42 . A method of screening to identify test substances which induce or repress the expression of genes which are induced or repressed by a protein or polypeptide of  claim 12  comprising: 
 (a) contacting a cell with a test substance; and  
 (b) monitoring expression of a transcript or its translation product, wherein the transcript specifically hybridizes to a gene selected from a first and a second group, wherein the first group consists of genes known to be transcriptionally upregulated by Rot: adaB, aldH, alsS, arcD, clfB, clpL, coa, ctpA, dhoM, dltB, dltC, dltD, dnax, epiA, fhuA, gltT, gtaB, guaA, guaB, hemn, hit, hld, holb, hsdR, isaB, lepA, lysP, lytH, lytR, lyts, mnha, msrA, mvaKl, nrdD, nrdE, nrdI, pbux, pth, purM, putp, pycA, recQ, rot, rpoc, sdrC, sigb, sodM, spa, srrB, thrb, thrC, xprT, and the second group consists of genes known to be transcriptionally downregulated by Rot: (hlb), adhe, cysK, ddh, ebhA, ebhB, fmhC(eprh), geh, gntK, gntp, hlgB, hlgC, kdpA, kdpC, lytN, mvaK2, mvaS, narG, pmi, prsA, ptsG, ribD, splA, splB, splC, splD, splE, splF, sspC, ureB, ureC, ureD, ureE, uref, ureG, wherein a test substance is identified if it increases expression of a transcript which specifically hybridizes to a gene in the first group or decreases expression of a transcript which specifically hybridizes to a gene in the second group.  
 
     
     
         43 . The method of  claim 42  wherein the transcript specifically hybridizes to a gene selected from the first group.  
     
     
         44 . The method of  claim 42  wherein the transcript specifically hybridizes to a gene selected from the second group.  
     
     
         45 . A diagnostic kit for detecting the presence of  Staphylococcus aureus  in a sample comprising: 
 (a) a pair of PCR primers, one member of the pair being a primer of  claim 27  and the other being a primer of  claim 28;     (b) a polymerase; and    (c) buffers and reagents for use in PCR.    
     
     
         46 . A diagnostic kit for detecting the presence of  Staphylococcus aureus  in a sample comprising: 
 (a) a pair of PCR primers, one member of the pair being a primer of  claim 29  and the other being a primer of  claim 30;     (b) a polymerase; and    (c) buffers and reagents suitable for use in PCR.    
     
     
         47 . A diagnostic kit for detecting the presence of a polynucleotide of  claim 2  in a microbial isolate or patient sample, comprising: 
 (a) a pair of PCR primers, one member of the pair being a primer of  claim 27  and the other being a primer of  claim 28;   
 (b) a polymerase; and  
 (c) buffers and reagents for use in PCR.  
 
     
     
         48 . A diagnostic kit for detecting the presence of a polynucleotide of  claim 3  in a microbial isolate or patient sample, comprising: 
 (a) a pair of PCR primers, one member of the pair being a primer of  claim 29  and the other being a primer of  claim 30;   
 (b) a polymerase; and  
 (c) buffers and reagents for use in PCR.  
 
     
     
         49 . A diagnostic kit for detecting the presence of a polynucleotide of  claim 2  in a microbial isolate or patient sample, comprising: 
 (a) an antibody of  claim 22  or  24 ; and  
 (b) one or more ancillary reagents for detecting the presence of a complex between said antibody and a polynucleotide.  
 
     
     
         50 . A diagnostic kit for detecting the presence of the polynucleotide as set forth in  claim 3  in a microbial isolate or patient sample, comprising: 
 (a) an antibody of  claim 23  or  25 ; and  
 (b) one or more ancillary reagents for detecting the presence of a complex between said antibody and a polynucleotide.  
 
     
     
         51 . A recombinant host cell comprising at least one copy of a recombinant vector of  claim 4  wherein said host cell or an ancestor of said host cell was transformed with said recombinant vector to produce said recombinant host cell, and wherein said nucleotide sequence is operably associated with an expression control sequence functional in said recombinant host cell.